Fluid shear stress from blood flow is a major determinant of vascular development, physiology and disease. In particular, atherosclerosis is initiated at sites of disturbed shear stress due to the inflammatory activation of endothelial cell. Past major advances from my lab are the identification of PECAM-1, VE-cadherin and VEGFR2 as a shear stress mechanosensor, and the discovery that the extracellular matrix under the endothelial cells determines whether flow signals are pro- or anti-inflammatory. Thus, basement membranes (in healthy vessels) suppress inflammation while fibronectin (deposited in inflamed, injured or remodeling vessels) permits inflammation. The current grant led to several advances. First, matrix specific effect are mediated by differential cAMP/protein kinase A signaling, which is high on basement membrane and low on fibronectin. This is mediated by binding of the main fibronectin receptor, integrin ?5?1, to phosphodiesterase 4D, which suppresses cAMP. Second, using our novel method to measure forces across proteins, we found that flow triggers an increase in force across PECAM-1, mediated by its de novo association with vimentin intermediate filaments, which transmit force from myosin. Third, we found that VE-cadherin functions as an adapter, binding VEGF receptors through its transmembrane domain, to promote its activation by flow. Fourth, we identified flow direction as an essential aspect of inflammatory activation of endothelial cells by disturbed flow, with flow parallel to the endothelil cytoskeletal axis activating anti- inflammatory pathways whereas perpendicular flow preferentially activates pro-inflammatory pathways. We also have new data linking VE-cadherin phosphorylation to polarity proteins in flow signaling. For the renewal, we propose: 1) To test the in vivo effects of altering extracellular matrix signaling by examining mice that contain mutations in the integrin ?5 subunit and in phosphodiesterase 4D that specifically block pro-inflammatory flow signaling. 2) To characterize the integrin-phosphodiesterase interaction in more detail. 3) To elucidate how flow direction determines signaling output. 4) To determine the consequences of mutating VE-cadherin on flow signaling in vivo. Together, these studies will lead advance our understanding of the molecular mechanisms by which flow acts upon endothelial cells and will test their in vivo consequences for blood vessel biology. Additionally, several aims have the potential to identify new drug targets for suppression of chronic inflammation including atherosclerosis.